Genetics of Indoxacarb resistance in Helicoverpa armigera (Hubner)

The present investigation was done with the aim of studying the genetics of Indoxacarb resistance. Selection of Helicoverpa armigera (Hubner) with Indoxacarb was done for eight generations to develop resistance. Reciprocal crosses between resistant and susceptible populations were made to understand the population genetics of Indoxacarb resistance in H. armigera . Generation-wise selection with Indoxacarb was evaluated for resistance development in H. armigera . The LC₅₀ of Indoxacarb was 2.81 p.p.m. for the first selected generation, and it increased to 272.55 p.p.m. after eight selected generations, which is a 1238.86-fold resistance compared to the susceptible strain. The estimated realized heritability (h²) after eight generations of selection with Indoxacarb was 0.45. The number of generations required for a tenfold increase in LC₅₀ (1/R) was estimated to be 2.59. The response to Indoxacarb selection in H. armigera was 0.39, the estimated selection differential (S) was 0.87, and the phenotypic standard deviation (σp) was 0.03. Reciprocal crosses between Indoxacarb resistant and susceptible strains revealed that the inheritance of Indoxacarb resistance was autosomal: neither maternal effect nor linkage was evident. The values of D_LC (0.10 and 0.09) indicated completely recessive inheritance of Indoxacarb resistance.     

Lethal and sub‐lethal effects of spinosad on the life‐history traits of army worm,Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae), and its fitness cost of resistance

Spodoptera litura is one of the most destructive polyphagous insect pests, with more than 120 host‐plant species. In our present study, a field‐collected population of S. litura when selected with spinosad for 11 consecutive generations resulted in the development of 3921‐fold resistance to spinosad as compared to the susceptible strain. The spinosad‐resistant strain of S. litura had a relatively high fitness cost (0.17) as compared to the susceptible strain. Furthermore, the lethal and sub‐lethal effects of different concentrations of spinosad were checked on the susceptible strain at different levels; i.e., LC₄₀, LC₃₀, LC₂₀ and LC₁₀, which revealed that the impact of spinosad on the life‐history traits of S. litura increased with the increase in concentration of spinosad. A significant impact of spinosad was recorded on the larval duration, pre‐pupal weight, pupal duration, pupal weight, reproductive potential and adult emergence. The outcomes of the current research clearly indicate that fitness cost of spinosad and its sub‐lethal effects have a significant impact on population dynamics of S. litura, for which it can be incorporated in integrated pest management.     

Field Evolved Resistance in Helicoverpa armigera (Lepidoptera: Noctuidae) to Bacillus thuringiensis Toxin Cry1Ac in Pakistan

Helicoverpa armigera (Hübner) is one of the most destructive pests of several field and vegetable crops, with indiscriminate use of insecticides contributing to multiple instances of resistance. In the present study we assessed whether H. armigera had developed resistance to Bt cotton and compared the results with several conventional insecticides. Furthermore, the genetics of resistance was also investigated to determine the inheritance to Cry1Ac resistance. To investigate the development of resistance to Bt cotton, and selected foliar insecticides, H. armigera populations were sampled in 2010 and 2011 in several cotton production regions in Pakistan. The resistance ratios (RR) for Cry1Ac, chlorpyrifos, profenofos, cypermethrin, spinosad, indoxacarb, abamectin and deltamethrin were 580-fold, 320-, 1110-, 1950-, 200-, 380, 690, and 40-fold, respectively, compared with the laboratory susceptible (Lab-PK) population. Selection of the field collected population with Cry1Ac in 2010 for five generations increased RR to 5440-fold. The selection also increased RR for deltamethrin, chlorpyrifos, profenofos, cypermethrin, spinosad, indoxacarb, abamectin to 125-folds, 650-, 2840-, 9830-, 370-, 3090-, 1330-fold. The estimated LC_50s for reciprocal crosses were 105 µg/ml (Cry1Ac-SEL female × Lab-PK male) and 81 g µg/ml (Lab-PK female × Cry1Ac-SEL male) suggesting that the resistance to Cry1Ac was autosomal; the degree of dominance (D_LC) was 0.60 and 0.57 respectively. Mixing of enzyme inhibitors significantly decreased resistance to Cry1Ac suggesting that the resistance to Cry1Ac and other insecticides tested in the present study was primarily metabolic. Resistance to Cry1Ac was probably due to a single but unstable factor suggesting that crop rotation with non-Bt cotton or other crops could reduce the selection pressure for H. armigera and improve the sustainability of Bt cotton.     

Resistance of sugarcane borer to Bacillus thuringiensis Cry1Ab toxin

The sugarcane borer, Diatraea saccharalis (F.) (Lepidoptera: Crambidae), strain (F52‐3‐R) was developed from F₃ survivors of a single‐pair mating on commercial Cry1Ab Bacillus thuringiensis (Bt) corn plants in the greenhouse. The susceptibility of a Bt‐susceptible and the F52‐3‐R strain of D. saccharalis to trypsin‐activated Cry1Ab toxin was determined in a laboratory bioassay. Neonate‐stage larvae were fed a meridic diet incorporating Cry1Ab toxin at a concentration range of 0.0625 to 32 µg g^?1. Larval mortality, larval weight, and number of surviving larvae that did not gain significant weight (<0.1 mg per larva) were recorded on the 7th day after inoculation. The F52‐3‐R strain demonstrated a significant level of resistance to the activated Cry1Ab toxin. Larval mortality of the Bt‐susceptible strain increased in response to higher concentrations of Cry1Ab toxin, exceeding 75% at 32 µg g^?1, whereas mortality of the F52‐3‐R strain was below 8% across all Cry1Ab concentrations. Using a measure of practical mortality (larvae either died or gained no weight), the median lethal concentration (LC₅₀) of the F52‐3‐R strain was 102‐fold greater than that of the Bt‐susceptible insects. Larval growth of both Bt‐susceptible and F52‐3‐R strains was inhibited on Cry1Ab‐treated diet, but the inhibition of the F52‐3‐R strain was significantly less than that of the Bt‐susceptible insects. These results confirm that the survival of the F52‐3‐R strain on commercial Bt corn plants was related to Cry1Ab protein resistance and suggest that this strain may have considerable value in studying resistance management strategies for Bt corn.     

Development and Characterization of Diamondback Moth Resistance to Transgenic Broccoli Expressing High Levels of Cry1C

A field-collected colony of the diamondback moth, Plutella xylostella, had 31-fold resistance to Cry1C protoxin of Bacillus thuringiensis. After 24 generations of selection with Cry1C protoxin and transgenic broccoli expressing a Cry1C protein, the resistance that developed was high enough that neonates of the resistant strain could complete their entire life cycle on transgenic broccoli expressing high levels of Cry1C. After 26 generations of selection, the resistance ratios of this strain to Cry1C protoxin were 12,400- and 63,100-fold, respectively, for the neonates and second instars by a leaf dip assay. The resistance remained stable until generation 38 (G38) under continuous selection but decreased to 235-fold at G38 when selection ceased at G28. The Cry1C resistance in this strain was seen to be inherited as an autosomal and incompletely recessive factor or factors when evaluated using a leaf dip assay and recessive when evaluated using Cry1C transgenic broccoli. Saturable binding of ¹²⁵I-Cry1C was found with brush border membrane vesicles (BBMV) from both susceptible and Cry1C-resistant strains. Significant differences in Cry1C binding to BBMV from the two strains were detected. BBMV from the resistant strain had about sevenfold-lower affinity for Cry1C and threefold-higher binding site concentration than BBMV from the susceptible strain. The overall Cry1C binding affinity was just 2.5-fold higher for BBMV from the susceptible strain than it was for BBMV from the resistant strain. These results suggest that reduced binding is not the major mechanism of resistance to Cry1C.     

Inheritance of evolved glyphosate resistance in <Emphasis Type="Italic">Conyza canadensis</Emphasis> (L.) Cronq.

N-(phosphonomethyl)glycine (glyphosate) resistance was previously reported in a horseweed [Conyza (=Erigeron) canadensis (L.) Cronq.] population from Houston, DE (P ₀ ^R ). Recurrent selection was performed on P ₀ ^R , since the population was composed of susceptible (5%) and resistant (95%) phenotypes. After two cycles of selection at 2.0 kg ae glyphosate ha^–1, similar glyphosate rates that reduced plant growth by 50%, glyphosate rates that inflicted 50% mortality in the population, and accumulations of half of the maximum detectable shikimic acid concentration were observed between the parental P ₀ ^R and the first (RS₁) and second (RS₂) recurrent generations. In addition, RS₁ and RS₂ did not segregate for resistance to glyphosate. This suggested that the RS₂ population comprised a near-homozygous, glyphosate-resistant line. Whole-plant rate responses estimated a fourfold resistance increase to glyphosate between RS₂ and either a pristine Ames, IA (P ₀ ^P ) or a susceptible C. canadensis population from Georgetown, DE (P ₀ ^S ). The genetics of glyphosate resistance in C. canadensis was investigated by performing reciprocal crosses between RS₂ and either the P ₀ ^P or P ₀ ^S populations. Evaluations of the first (F₁) and second (F₂) filial generations suggested that glyphosate resistance was governed by an incompletely dominant, single-locus gene (R allele) located in the nuclear genome. The proposed genetic model was confirmed by back-crosses of the F₁ to plants that arose from achenes of the original RS₂, P ₀ ^P , or P ₀ ^S parents. The autogamous nature of C. canadensis, the simple inheritance model of glyphosate resistance, and the fact that heterozygous genotypes (F₁) survived glyphosate rates well above those recommended by the manufacturer, predicted a rapid increase in frequency of the R allele under continuous glyphosate selection. The impact of genetics on C. canadensis resistance management is discussed.     

Inheritance of Bacillus thuringiensis Cry1C resistance in Egyptian cotton leafworm,Spodoptera littoralis (Lepidoptera: Noctuidae)

A field strain of Spodoptera littoralis Biosduval was selected against Cry1C toxin derived from Bacillus thuringiensis entomocidus for 10 subsequent generations under laboratory conditions. Selection pressure resulted in a 29‐fold resistance ratio compared with the susceptible strain. Inheritance of Cry1C resistance was partially dominant and autosomal on the basis of bioassay response to Cry1C toxin in a reciprocal cross between male and/or female F₁. Consistent with earlier findings, resistance was recessive at high concentrations of Cry1C toxin. However, the dominance of resistance increased as the concentration of Cry1C decreased. Analysis of survival and growth of progeny from a backcross (F₁ × resistance strain) suggested that resistance was controlled by either a single or a few loci in cotton leafworm.     

Transgenic Rice Plants Expressing a Modified <Emphasis Type="Italic">cry1Ca1</Emphasis> Gene are Resistant to <Emphasis Type="Italic">Spodoptera litura</Emphasis> and <Emphasis Type="Italic">Chilo</Emphasis><Emphasis Type="Italic">suppressalis</Emphasis>

Nucleotide sequence encoding the truncated insecticidal Cry1Ca1 protein from Bacillus thuringiensis was extensively modified based on the codon usage of rice genes. The overall G + C contents of the synthetic cry1Ca1 coding sequence were raised to 65% with an additional bias of enriching for G and C ending codons as preferred by monocots. The synthetic gene was introduced into the Chinese japonica variety, Xiushui 11, by Agrobacterium-mediated transformation. Transgenic rice plants harboring this gene were highly resistant to Chilo suppressalis and Spodoptera litura larvae as revealed by insect bioassays. High levels of Cry1Ca1 protein were obtained in the leaves of transgenic rice, which were effective in achieving 100% mortality of S. litura and C. suppressalis larvae. The levels of Cry1Ca1 expression in the leaves of these transgenic plants were up to 0.34% of the total soluble proteins. The larvae of C. suppressalis and S. litura could consume a maximum of 1.89  and 4.89 mm² of transgenic leaf area whereas the consumption of non-transgenic leaves by these larvae was significantly higher; 58.33 and 61.22 mm², respectively. Analysis of R₁ transgenic plants indicated that the cry1Ca1 was inherited by the progeny plants and provided complete protection against C. suppressalis and S. litura larvae.     

Highly Toxic and Broad-Spectrum Insecticidal Bacillus thuringiensis Engineered by Using the Transposon Tn917 and Protoplast Fusion

The chromosome of the Bacillus thuringiensis strain S184 that was toxic against the third instar larvae of Spodoptera litura with the LC₅₀ of 9.74 μg/ml was successfully integrated into two genes of cyt1Aa and cry11Aa using the transposon Tn917, yielding the primary engineered strain TnX. The strain TnX was highly toxic to the third instar larvae of Culex pipiens fatigans with the LC₅₀ of 5.12 ng/ml which was 1.82-fold higher than that of B. thuringiensis subsp. israelensis, but lowly toxic to lepidopterous larvae. By the protoplast fusion of the strain TnX and the strain S184-Tet^r (resistance to tetracycline), the target engineered strain TnY was obtained. Against the third instar larvae of S. litura, the strain TnY LC₅₀ was of 4.68 μ g/ml and increased by 2.08-fold in comparison with the parent strain S184. Against the third instar larvae of C. pipiens fatigans, the strain TnY LC₅₀ was of 103.20 ng/ml. The two target genes of cyt1Aa and cry11Aa integrated into the chromosome were extremely stable and had little possibility of a second transposition. It was unclear whether some factors existing in the parent strain, S184, contributed to the high toxicity of the strains TnX and TnY. Received: 30 November 2000 / Accepted: 10 January 2001     

Inheritance of Resistance to the Bacillus thuringiensis Toxin Cry1C in the Diamondback Moth

Laboratory selection increased resistance to the Bacillus thuringiensis toxin Cry1C in a strain of diamondback moth (Plutella xylostella). The selected strain was derived from a field population that had evolved high levels of resistance to Bacillus thuringiensis subsp. kurstaki and moderate resistance to Cry1C. Relative to the responses of a susceptible strain of diamondback moth, the resistance to Cry1C of the selected strain increased to 62-fold after six generations of selection. The realized heritability of resistance was 0.10. Analysis of F(inf1) hybrid progeny from reciprocal crosses between the selected strain and a susceptible strain showed that resistance to Cry1C was autosomally inherited. The dominance of resistance to Cry1C depended on the concentration; inheritance was increasingly dominant as the concentration decreased. Responses of progeny from single-pair families showed that resistance to Cry1C and resistance to Cry1Ab were inherited independently, which enhances opportunities for managing resistance. However, compared with projections based on previously reported recessive inheritance of resistance to Cry1A toxins, the potentially dominant inheritance of resistance to Cry1C observed here could accelerate evolution of resistance.     

棉蚜对吡虫啉的抗性选育和现实遗传力分析

【目的】为了评估棉蚜Aphis gossypii Glover对吡虫啉的抗性风险,在室内进行了棉蚜对吡虫啉(imidacloprid)的抗性选育和抗性现实遗传力分析。【方法】采用单头反选育法和群体汰选法,分别得到了棉蚜对吡虫啉敏感品系(LC₅₀为0.176 mg/L)和抗性品系(LC₅₀为14.657 mg/L)。采用阈性状分析方法,获得棉蚜对吡虫啉的抗性现实遗传力（h²）。【结果】相对于田间原始种群(LC₅₀为0.346 mg/L),吡虫啉敏感棉蚜品系对吡虫啉的LC50减少了2倍;获得的吡虫啉抗性棉蚜品系,经过40代的选育,得到抗性倍数为室内敏感品系的83.27倍的抗性品系。棉蚜对吡虫啉的抗性现实遗传力(h2)为0.1478。进一步预测其抗性发展速度,基于80%~90%的选择压力,预计抗性增长100倍时,吡虫啉可使用30.2~38.1代。【结论】这些研究说明棉蚜对吡虫啉存在抗性风险。     

Fitness costs of Cry1F resistance in fall armyworm,Spodoptera frugiperda

Transgenic corn, Zea mays L., expressing the Bacillus thuringiensis Berliner (Bt) protein Cry1F has been registered for Spodoptera frugiperda (J. E. Smith) control since 2003 in the USA. Unexpected damage to Cry1F corn was reported in 2006 in Puerto Rico, and Cry1F resistance in S. frugiperda from Puerto Rico was documented. The study of fitness costs associated with insect resistance to Bt insecticidal proteins is important for understanding resistance evolution and for evaluating resistance management practices used to mitigate resistance to transgenic corn. Currently, no studies have addressed the fitness costs associated with Cry1F resistance in S. frugiperda. In this study, susceptible and resistant strains with similar genetic background and their reciprocal crosses were used to estimate Cry1F resistance fitness costs. Comparisons between life‐history traits and population growth rates of homozygous susceptible, heterozygous and homozygous resistant S. frugiperda were used to determine whether the resistance is associated with fitness costs. Major fitness costs were not apparent in either heterozygotes or homozygous resistant insects. However, there was a slight indication of hybrid vigour in the heterozygotes. Additionally, two lines in which the frequency of the resistant alleles was fixed at 0.5 were followed for seven generations, after which the frequency of resistant alleles slightly decreased in both lines. The lack of strong fitness costs associated with Cry1F resistance in S. frugiperda indicates that initial allele frequencies may be higher than expected in field populations and will tend to remain stable in field populations in the absence of selection pressure (e.g. Puerto Rico).     

Effects of Bacillus thuringiensis Cry1C toxin on the metabolic rate of Cry1C resistant and susceptible Spodoptera exigua (Lepidoptera: Noctuidae)

Abstract. The effects of Bacillus thuringiensis (Bt) Cry1C toxin on the metabolic rate of Cry1C resistant and susceptible Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae) are investigated using closed‐system respirometry. Mechanisms of resistance to the Bt toxin may be associated with an energetic cost that can be measured as an increase in metabolic rate compared with Bt‐susceptible insects. This hypothesis is tested using third‐ and fifth‐instar larvae and 1–7‐day‐old pupae. Metabolic rate is measured as the amount of O₂ consumed and CO₂ produced. V?_O2 and V?_CO2 (mL g^?1 h^?1) of third‐instar Cry1C resistant larvae reared continuously on a diet containing 320 µg Cry1C toxin per g diet (CryonT) are significantly greater than third‐instar Cry1C resistant larvae reared on toxin for 5 days and reared thereafter on untreated diet (Cry5dT), Cry1C resistant larvae reared on untreated diet (CryReg) and the susceptible parental strain (SeA) reared on untreated diet. There are no differences in V?_O2 and V?_CO2 (mL g^?1 h^?1) among treatment groups for fifth‐instar larvae. CryonT larvae and pupae weigh significantly less than larvae and pupae receiving other treatments. Smaller body mass may be an important biological cost to individuals exposed continuously to Bt toxin. One‐day‐old pupae of all treatment groups exhibit a high V?_O2 (mean approximately 0.174 mL g^?1 h^?1) with CryonT having a significantly greater value than all other treatments; there are no differences among the other treatments. Pupal metabolic rates of all treatment groups decline to a minimum between days 2 and 4 then increase linearly between days 4 and 7 until adult emergence. These results demonstrate no difference in metabolic rates, and possibly fitness costs, between resistant (CryReg and Cry5dT) and susceptible (SeA) S. exigua except when larvae were reared continuously on toxin (CryonT).     

Inheritance of resistance to Bacillus thuringiensis Cry1Ab protein in the sugarcane borer (Lepidoptera: Crambidae)

Inheritance traits of a Cry1Ab-resistant strain of the sugarcane borer, Diatraea saccharalis (F.) were analyzed using various genetic crosses. Reciprocal parental crosses between Cry1Ab-susceptible and Cry1Ab-resistant populations, F₁ by F₁ crosses, and backcrosses of F₁ with the Cry1Ab-resistant population were successfully completed. Larval mortality of the parental and cross-populations were assayed on Cry1Ab diet and Bacillus thuringiensis (Bt)-corn leaf tissue. Maternal effects and sex linkage were examined by comparing the larval mortality between the two F₁ populations. Dominance levels of resistance were measured by comparing the larval mortality of the Cry1Ab-resistant, -susceptible, and -heterozygous populations. Number of genes associated with the resistance was evaluated by fitting the observed mortality of F₂ and backcross populations with a Mendelian monogenic inheritance model. Cry1Ab resistance in D. saccharalis was likely inherited as a single or a few tightly linked autosomal genes. The resistance was incompletely recessive on Bt corn leaf tissue, while the effective dominance levels (D_ML) of resistance increased as Cry1Ab concentrations decreased with Cry1Ab-treated diet. D_ML estimated based on larval mortality on intact Bt corn plants reported in a previous study ranged from 0.08 to 0.26. This variability in D_ML levels of Cry1Ab resistance in D. saccharalis suggests that Bt corn hybrids must express a sufficient dose of Bt proteins to make the resistance genes functionally recessive. Thus, Bt resistant heterozygous individuals can be killed as desired in the “high/dose refuge” resistance management strategy for Bt corn.     

Rapid selection and characterization of Cry1F resistance in a Brazilian strain of fall armyworm

Transgenic maize (Zea mays L., Poaceae) event TC1507, producing the Cry1F protein of Bacillus thuringiensis Berliner, has been used for management of the fall armyworm, Spodoptera frugiperda (JE Smith) (Lepidoptera: Noctuidae), in Brazil since 2009. A strain of S. frugiperda, obtained from field collections of larvae in TC1507 maize in Minas Gerais state in 2010, was selected in the laboratory for resistance to Cry1F using leaves of TC1507 maize in two selection regimes. Continuous exposure of larvae to Cry1F was more effective than exposure for 6, 8, and 10 days in the selection of resistant S. frugiperda individuals. With only four generations of laboratory selection, a strain with high levels of resistance to Cry1F was obtained, as indicated by the survival of insects reared on leaves of TC1507 maize plants and by the more than 300‐fold resistance level measured in bioassays with the purified Cry1F protein. Importantly, reciprocal crosses between control and the Cry1F‐selected strains revealed that the resistance is autosomal and incompletely recessive, and the response obtained in the backcross of the F₁ generation with the resistant strain was consistent with simple monogenic inheritance. Additionally, there were no apparent fitness costs associated with resistance either for survival or larval growth on non‐Bt maize leaves. Our findings provide experimental evidence for rapid evolution of Cry1F resistance in S. frugiperda in the laboratory and further reinforce the potential of this species to evolve field resistance to the TC1507 maize as previously reported. The resistant strain isolated in this study provides an opportunity to estimate the resistance allele frequency in the field and to determine the biochemical and molecular basis of the resistance, which should provide further information to assist in the resistance management of S. frugiperda on transgenic maize producing B. thuringiensis proteins.     

Characterization of resistance to Bacillus thuringiensis toxin Cry1Ac in Plutella xylostella from China

A field population (SZ) of Plutella xylostella, collected from the cabbage field in Shenzhen, Guangdong Province of China in 2002, showed 2.3-fold resistance to Cry1Aa, 110-fold to Cry1Ab, 30-fold to Cry1Ac, 2.1-fold to Cry1F, 5.3-fold to Cry2Aa and 6-fold resistance to Bacillus thuringiensis var. kurstaki (Btk) compared with a susceptible strain (ROTH). The SZBT strain was derived from the SZ population through 20 generations of selection with activated Cry1Ac in the laboratory. While the SZBT strain developed 1200-fold resistance to Cry1Ac after selection, resistance to Cry1Aa, Cry1Ab, Cry1F, and Btk increased to 31-, 1900-,>33- and 17-fold compared with the ROTH strain. However, little or no cross-resistance was detected to Cry1B, Cry1C and Cry2Aa in the SZBT strain. Genetic cross analyses between the SZBT and ROTH strains revealed that Cry1Ac-resistance in the SZBT strain was controlled by a single, autosomal, incompletely recessive gene. Binding studies with ¹²⁵I-labeled Cry1Ac showed that the brush border membrane vesicles (BBMVs) of midguts from the resistant SZBT insects had lost binding to Cry1Ac. Allelic complementation tests demonstrated that the major Bt resistance locus in the SZBT strain was same as that in the Cry1Ac-R strain which has “mode 1” resistance to Bt. An F₁ screen of 120 single-pair families between the SZBT strain and three field populations collected in 2008 was carried out. Based on this approach, the estimated frequencies of Cry1Ac-resistance alleles were 0.156 in the Yuxi population from Yunnan province, and 0.375 and 0.472 respectively in the Guangzhou and Huizhou populations from Guangdong province.     

Development of a hybrid <Emphasis Type="Italic">delta</Emphasis>-endotoxin and its expression in tobacco and cotton for control of a polyphagous pest <Emphasis Type="Italic">Spodoptera litura</Emphasis>

A hybrid -endotoxin protein was designed against a polyphagous lepidopteran insect pest Spodoptera litura, which is tolerant to most of the known -endotoxins. The hybrid -endotoxin was created by replacing amino acid residues 530–587 in a poorly active natural Cry1Ea protein, with a highly homologous 70 amino acid region of Cry1Ca in domain III. The truncated -endotoxins Cry1Ea, Cry1Ca and the hybrid protein Cry1EC accumulated in Escherichia coli to form inclusion bodies. The solubilised Cry1EC made from E. coli was 4- fold more toxic to the larvae of S. litura than Cry1Ca, the best known -endotoxin against Spodoptera sp. None of the two truncated toxins, solubilised from E. coli caused larval mortality. However, trypsinised Cry1Ca protoxin obtained from E. coli and solubilised from inclusion bodies caused mortality of S. litura with LC₅₀ 513 ng/ml semi synthetic diet. A synthetic gene coding for the hybrid$-endotoxin Cry1EC was designed for high level expression in plants, taking into consideration several features found in the highly expressed plant genes. Transgenic, single copy plants of tobacco as well as cotton were developed. The selected lines expressed Cry1EC at 0.1–0.7% of soluble leaf protein. Such plants were completely resistant to S. litura and caused 100% mortality in all stages of larval development. Hence, unlike in E. coli, the hybrid -endotoxin folded into a functionally active conformation in both tobacco and cotton leaves. The truncated Cry1EC expressed in tobacco leaves was about 8-fold more toxic (LC₅₀ 58 ng/ml diet) compared to expression in E. coli.     

Biochemical mechanism of chlorantraniliprole resistance in Spodoptera litura (Fab) (Lepidoptera: Noctuidae)

Spodoptera litura, a polyphagus insect pest of economic importance, having the ability to develop resistance to various classes of insecticides was selected for the study. Leaf dip bioassay studies were done after ten generations of selection pressure reported the development of resistance up to 80 folds by S. litura against chlorantraniliprole compared to lab susceptible strain. Bioassay studies conducted using enzyme inhibitors such as triphenyl phosphate (TPP) followed by diethyl-maleate (DEM) and piperonyl butoxide (PBO) showed good amount of synergism with chlorantraniliprole and improved efficacy against resistant strain. Findings of bioassay studies were supported by in-vitro enzyme inhibition assays. Esterase activity in gut homogenate of resistant strain was significantly inhibited by TPP suggesting esterase mediated biochemical resistance development in S. litura against chlorantraniliprole.     

Inheritance of resistance to leaf and glume blotch caused by Septoria nodorum Berk. in winter wheat

Sixteen crosses between eight winter wheat cultivars were screened for resistance to Septoria nodorum leaf and glume blotch in the F₁ and F₄ generations using artificial inoculation in the field. The F₁ of most crosses showed dominance for susceptibility on both ear and leaf. The effects of general combining ability were of similar magnitude as the effects for specific combining ability. On the basis of the phenotypic difference of the parents, no prediction was possible about the amount and the direction of genetic variance in the segregating populations. The variation observed in this study both within and among the segregating populations suggests a quantitative inheritance pattern influencing the expression of the two traits. The components of variance between F₂ families within a population were as high as (for S. nodorum blotch on the ear) or higher (for S. nodorum blotch on the leaf) than those between populations. Therefore, strong selection within a few populations may be as effective to obtain new resistant genotypes as selection in a large number of populations. In almost all crosses, progenies were found that were more resistant than the better parent. Thus transgression breeding may be a tool to breed for higher levels of resistance to S. nodorum blotch. Highly resistant genotypes were found even in combination with two susceptible parents. The genetic source for Septoria resistance is probably broader than is generally assumed and could be used to improve S. nodorum resistance by combination breeding followed by strong selection in large populations. Received: 18 January / Accepted: 30 April 1999     

Expression of the cry1EC gene in castor (Ricinus communis L.) confers field resistance to tobacco caterpillar (Spodoptera litura Fabr) and castor semilooper (Achoea janata L.)

Castor (cv. DCS-9) has been transformed through Agrobacterium-mediated and particle gun bombardment methods using appropriate vectors containing the Bt chimeric gene cry1EC driven by enhanced 35S promoter. About 81 and 12 putative transformants were regenerated following selection on hygromycin and kanamycin, respectively. Southern analysis of DNA extracted from T₀ plants confirmed integration of the introduced gene in castor genome. The integration and inheritance of the introduced genes was demonstrated up to T₄ generation by PCR and Southern analysis. Southern analysis of two events having single and two copies showed the same pattern of integration in the subsequent generations. Insect feeding experiments conducted in the laboratory by releasing neonate larvae of castor semilooper and S. litura on leaf tissues excised from transgenic and control plants showed varying degrees of larval mortality and slow growth in larvae fed on transgenic leaf tissue. Field bioassays against Spodoptera litura and castor semilooper conducted for eight events in T₁–T₄ generations under net confinement were more informative and events conferring resistance to the two major defoliators were identified.